title: Lyssavirus detection and typing using pyrosequencing.
abstract: Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3' terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.
authors.affiliation: OIE and National Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro, Padova, Italy. pdebenedictis@izsvenezie.it
authors.forename: Paola
authors.initials: P
authors.lastname: De Benedictis
authors.forename: Cristian
authors.initials: C
authors.lastname: De Battisti
authors.forename: Laurent
authors.initials: L
authors.lastname: Dacheux
authors.forename: Sabrina
authors.initials: S
authors.lastname: Marciano
authors.forename: Silvia
authors.initials: S
authors.lastname: Ormelli
authors.forename: Angela
authors.initials: A
authors.lastname: Salomoni
authors.forename: Silvia Tiozzo
authors.initials: ST
authors.lastname: Caenazzo
authors.forename: Anthony
authors.initials: A
authors.lastname: Lepelletier
authors.forename: Hervé
authors.initials: H
authors.lastname: Bourhy
authors.forename: Ilaria
authors.initials: I
authors.lastname: Capua
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cattoli
mesh_terms: D000818:Animals; D006801:Humans; D009698:Nucleoproteins / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011818:Rabies / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D011820:Rabies virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
chemical_list: D009698:Nucleoproteins; D012367:RNA, Viral; D014764:Viral Proteins